Combating herpesvirus encephalitis by potentiating a TLR3-mTORC2 axis.

TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly 'rescued' by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3-mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis.

Structural basis of CXC chemokine receptor 2 activation and signalling.

Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor-chemokine recognition2-4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles.

Selenium-SelK-GPX4 axis protects nucleus pulposus cells against mechanical overloading-induced ferroptosis and attenuates senescence of intervertebral disc.

Intervertebral disc degeneration (IVDD) is one of the most prevalent spinal degenerative disorders and imposes places heavy medical and economic burdens on individuals and society. Mechanical overloading applied to the intervertebral disc (IVD) has been widely recognized as an important cause of IVDD. Mechanical overloading-induced chondrocyte ferroptosis was reported, but the potential association between ferroptosis and mechanical overloading remains to be illustrated in nucleus pulposus (NP) cells. In this study, we discovered that excessive mechanical loading induced ferroptosis and endoplasmic reticulum (ER) stress, which were detected by mitochondria and associated markers, by increasing the intracellular free Ca2+ level through the Piezo1 ion channel localized on the plasma membrane and ER membrane in NP cells. Besides, we proposed that intracellular free Ca2+ level elevation and the activation of ER stress are positive feedback processes that promote each other, consistent with the results that the level of ER stress in coccygeal discs of aged Piezo1-CKO mice were significantly lower than that of aged WT mice. Then, we confirmed that selenium supplementation decreased intracellular free Ca2+ level by mitigating ER stress through upregulating Selenoprotein K (SelK) expression. Besides, ferroptosis caused by the impaired production and function of Glutathione peroxidase 4 (GPX4) due to mechanical overloading-induced calcium overload could be improved by selenium supplementation through Se-GPX4 axis and Se-SelK axis in vivo and in vitro, eventually presenting the stabilization of the extracellular matrix (ECM). Our findings reveal the important role of ferroptosis in mechanical overloading-induced IVDD, and selenium supplementation promotes significance to attenuate ferroptosis and thus alleviates IVDD, which might provide insights into potential therapeutic interventions for IVDD.

Development of Novel Silicon Quantum Dots and Their Potential in Improving the Enhanced Oil Recovery of HPAM.

Hydrolyzed polyacrylamide (HPAM) is commonly used in polymer flooding, however, it is prone to viscosity reduction at high temperatures and high salinities, weakening its ability to improve oil recovery. In this work, sulfonated modified silicon quantum dots (S-SiQDs) were synthesized and then added to HPAM to study the improvement of rheological properties and enhanced oil recovery performance of HPAM at high temperatures and salinities. It is found that the S-SiQDs with a concentration of only 0.1 wt % can significantly increase the viscosity of HPAM from 28.5 to 39.6 mPa·s at 60 °C and 10,000 mg/L NaCl. Meanwhile, the HPAM/S-SiQDs hybrid solution always possessed higher viscosity and viscoelastic moduli than HPAM, attributed to the hydrogen bonding between HPAM and S-SiQDs. Notably, HPAM/S-SiQDs still maintained elastic behavior at harsh conditions, indicating that they formed a strong network structure. Through oil displacement experiments, it was found that the oil recovery of HPAM/S-SiQDs was higher (28.3%), while that of HPAM was only 17.2%. Thereafter, the utilization sequence of oil during the displacement process was studied with nuclear magnetic resonance experiments. Ultimately, the oil displacement mechanism of HPAM/S-SiQDs was deeply analyzed, including viscosity thickening and wetting reversal.

Microwave ablation versus surgical resection for subcapsular hepatocellular carcinoma: a propensity score-matched study of long-term therapeutic outcomes.

OBJECTIVES: The therapeutic efficacy of microwave ablation (MWA) for subcapsular hepatocellular carcinoma (HCC) has not been well characterized. We aimed to compare the long-term outcomes of MWA and surgical resection (SR) in patients with subcapsular HCC. METHODS: This retrospective study comprised 321 patients with subcapsular HCC meeting the Milan criteria who received MWA (n = 99) or SR (n = 222). Local tumor progression (LTP), overall survival (OS), and disease-free survival (DFS) were analyzed using propensity score matching (PSM) to compare the therapeutic efficacy. RESULTS: In the total cohort, there were no significant differences in 5-year LTP rates (14.0% vs. 8.9%, p = 0.12), OS rates (70.7% vs. 73.2%, p = 0.63), and DFS rates (38.3% vs. 41.2%, p = 0.22) between the MWA and SR groups. After PSM, the cumulative LTP rates at 1, 3, and 5 years were 9.7%, 14.0%, and 16.4% in the MWA group (n = 84) and 7.2%, 8.6%, and 10.6% in the SR group (n = 84), respectively, with no significant difference (p = 0.31). Neither corresponding OS rates (96.4%, 84.8%, and 73.0% vs. 95.2%, 85.5%, and 72.1%, p = 0.89) nor DFS rates (76.0%, 52.6%, and 38.1% vs. 76.2%, 44.7%, and 32.3%, p = 0.43) were significantly different between the MWA and SR groups. Whereas MWA obtained fewer complications for both cohorts (both p < 0.05). CONCLUSION: MWA showed comparable long-term therapeutic outcomes to SR, and it might be an alternative curative option for subcapsular HCC within the Milan criteria. KEY POINTS: • Microwave ablation showed comparable local tumor progression, overall survival, and disease-free survival to surgical resection for subcapsular hepatocellular carcinoma meeting the Milan criteria. • Microwave ablation obtained fewer complications and shorter postoperative hospital stay.

Structures and bonding properties of lithium polysulfide clusters LiSn-/0 (n = 3-5) and Li2S4-/0: size-selected anion photoelectron spectroscopy and theoretical calculations.

The structures and bonding properties of several lithium polysulfide clusters LiSn-/0 (n = 3-5) and Li2S4-/0 were investigated by size-selected anion photoelectron spectroscopy coupled with quantum chemistry calculations. The vertical detachment energies of LiS3-, LiS4-, and LiS5- were estimated to be 2.17 ± 0.08, 3.30 ± 0.08 and 3.66 ± 0.08 eV, respectively, and that of Li2S4- was estimated to be 3.21 ± 0.08 eV. It is found that LiS3- and LiS3 have planar quadrilateral structures, and LiS4- and LiS4 have distorted five-membered ring structures. LiS5- has a distorted six-membered ring structure while neutral LiS5 has a book-shaped structure. The lowest-lying structure of Li2S4- can be viewed as a S2 unit connecting to the Li-Li edge of a Li2S2 tetrahedron. The lowest-lying structure of neutral Li2S4 can be viewed as a S2 unit connecting to the S atoms of a Li2S2 quadrilateral. The natural population analysis (NPA) and electron localization function (ELF) analyses show that the excess electron of LiSn- is mainly localized over the sulfur chains, especially on the S atoms interacting with Li, thus, the most stable structures of LiSn- can be regarded as a Li+ cation interacting with a Sn2- dianion. The results may be useful for understanding the formation of lithium polysulfides in lithium sulfur batteries.

Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators.

The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR and located outside helices V-VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

Dynamic landscape and regulation of RNA editing in mammals.

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Skewed endosomal RNA responses from TLR7 to TLR3 in RNase T2-deficient macrophages.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.

Microwave ablation versus radiofrequency ablation for subcapsular hepatocellular carcinoma: a propensity score-matched study.

OBJECTIVES: Thermal ablation is now accepted as one of the curative treatments for patients with early-stage hepatocellular carcinoma (HCC), but the efficacy of this treatment for subcapsular HCC is not well characterized. Therefore, we aimed to compare the outcomes of microwave ablation (MWA) and radiofrequency ablation (RFA) for patients with subcapsular HCC. METHODS: In total, 195 patients with subcapsular HCC who met the Milan criteria and underwent MWA or RFA were included. Local tumor progression (LTP), overall survival (OS), recurrence beyond the Milan criteria (RBM), and complications of these patients were compared. RESULTS: After propensity score matching, the 1-, 3-, and 5-year cumulative LTP rates were 6.7%, 9.6%, and 11.4% in the MWA group, and 13.4%, 24.6%, and 29.1% in the RFA group, respectively (p = 0.006). The cumulative rates of RBM were lower in patients treated with MWA than in those treated with RFA (4.4% versus 12% at 1 year; 14.5% versus 23.0% at 3 years; and 37.4% versus 53.9% at 5 years; p = 0.03). The OS rates at 1, 3, and 5 years were 97.1%, 85.9%, and 73.4% in the MWA group, and 95.6%, 80.4%, and 61.4% in the RFA group, respectively (p = 0.36). The rate of major complications showed no significant difference between the MWA group and the RFA group (17.4% vs. 11.6%, p = 0.33). CONCLUSION: Compared to RFA, MWA showed better tumor control for subcapsular HCC within the Milan criteria. There was no difference in the incidence of major complications between the two groups. KEY POINTS: •Compared to radiofrequency ablation, microwave ablation showed better local tumor control for patients with subcapsular hepatocellular carcinoma. •Microwave ablation showed similar major complication rates for patients with subcapsular hepatocellular carcinoma. •Microwave ablation may be preferred for patients with subcapsular hepatocellular carcinoma when they need to receive thermal ablation.

Genetic Diversity Analysis Reveals Potential of the Green Peach Aphid (Myzus persicae) Resistance in Ethiopian Mustard.

Brassica carinata (BBCC, 2n = 34) is commonly known as Ethiopian mustard, Abyssinian mustard, or carinata. Its excellent agronomic traits, including resistance to biotic and abiotic stresses, make it a potential genetic donor for interspecific hybridization. Myzus persicae (green peach aphid, GPA) is one of the most harmful pests of Brassica crops, significantly effecting the yield and quality. However, few aphid-resistant Brassica crop germplasms have been utilized in breeding practices, while the underlying biochemical basis of aphid resistance still remains poorly understood. In this study, we examined the genetic diversity of 75 B. carinata accessions and some plant characteristics that potentially contribute to GPA resistance. Initially, the morphological characterization showed abundant diversity in the phenotypic traits, with the dendrogram indicating that the genetic variation of the 75 accessions ranged from 0.66 to 0.98. A population structure analysis revealed that these accessions could be grouped into two main subpopulations and one admixed group, with the majority of accessions (86.67%) clustering in one subpopulation. Subsequently, there were three GPA-resistant B. carinata accessions, BC13, BC47, and BC51. The electrical penetration graph (EPG) assay detected resistance factors in the leaf mesophyll tissue and xylem. The result demonstrated that the Ethiopian mustard accessions were susceptible when the phloem probing time, the first probe time, and the G-wave time were 20.51-32.51 min, 26.36-55.54 s, and 36.18-47.84 min, respectively. In contrast, resistance of the Ethiopian mustard accessions was observed with the phloem probing time, the first probe time, and G-wave time of 41.18-70.78 min, 181.07-365.85 s, and 18.03-26.37 min, respectively. In addition, the epidermal characters, leaf anatomical structure, glucosinolate composition, defense-related enzyme activities, and callose deposition were compared between the resistant and susceptible accessions. GPA-resistant accessions had denser longitudinal leaf structure, higher wax content on the leaf surface, higher indole glucosinolate level, increased polyphenol oxidase (PPO) activity, and faster callose deposition than the susceptible accessions. This study validates that inherent physical and chemical barriers are evidently crucial factors in the resistance against GPA infestation. This study not only provide new insights into the biochemical basis of GPA resistance but also highlights the GPA-resistant B. carinata germplasm resources for the future accurate genetic improvement of Brassica crops.

TNF-α induces up-regulation of MicroRNA-27a via the P38 signalling pathway, which inhibits intervertebral disc degeneration by targeting FSTL1.

The mechanism of intervertebral disc degeneration is still unclear, and there are no effective therapeutic strategies for treating this condition. miRNAs are naturally occurring macromolecules in the human body and have many biological functions. Therefore, we hope to elucidate whether miRNAs are associated with intervertebral disc degeneration and the underlying mechanisms involved. In our study, differentially expressed miRNAs were predicted by the GEO database and then confirmed by qPCR and in situ hybridization. Apoptosis of nucleus pulposus cells was detected by flow cytometry and Bcl2, Bax and caspase 3. Deposition of extracellular matrix was assessed by Alcian blue staining, and the expression of COX2 and MMP13 was detected by immunofluorescence, Western blot and qPCR. Moreover, qPCR was used to detect the expression of miR27a and its precursors. The results showed that miR27a was rarely expressed in healthy intervertebral discs but showed increased expression in degenerated intervertebral discs. Ectopic miR27a expression inhibited apoptosis, suppressed the inflammatory response and attenuated the catabolism of the extracellular matrix by targeting FSTL1. Furthermore, it seems that the expression of miR27a was up-regulated by TNF-α via the P38 signalling pathway. So we conclude that TNF-α and FSTL1 engage in a positive feedback loop to promote intervertebral disc degeneration. At the same time, miR27a is up-regulated by TNF-α via the P38 signalling pathway, which ameliorates inflammation, apoptosis and matrix degradation by targeting FSTL1. Thus, this negative feedback mechanism might contribute to the maintenance of a low degeneration load and would be beneficial to maintain a persistent chronic disc degeneration.

The impact of cell maturation and tissue microenvironments on the expression of endosomal Toll-like receptors in monocytes and macrophages.

Toll-like receptors (TLRs) impact myeloid cell responsiveness to environmental cues such as pathogen components and metabolites. Although TLR protein expression in monocytes and tissue macrophages is thought to be optimized for microenvironments in each tissue, a comprehensive study has not been reported. We here examined protein expression of endogenous TLRs in tissue-resident myeloid cells. Neutrophils in peripheral blood, spleen, liver and lung expressed TLR2, TLR4 and TLR5 in all tissues. Ly6C+ MHC II‒ classical monocytes mature into Ly6C‒ MHC II+ monocyte-derived dendritic cells (moDCs) or Ly6C‒ MHC II‒ patrolling monocytes. These subsets were found in all the tissues studied. TLR2 and TLR4 were displayed on all of these subsets, regardless of location. In contrast, expression of endosomal TLRs did vary with tissues and subsets. moDCs expressed TLR9, but much less TLR7. In contrast, TLR7, not TLR3 or TLR9, was highly expressed in classical and patrolling monocytes. Tissue macrophages such as red pulp macrophages in the spleen, Kupffer cells in the liver, microglia in the brain, alveolar macrophages in the lung and adipose tissue macrophages all expressed TLR2, TLR4 and TLR3. TLR7 was also expressed in these tissue macrophages except Kupffer cells in the liver. TLR9 expression in tissue macrophages was much lower or hard to detect. These results suggest that expression of endosomal TLRs in myeloid cells is influenced by their differentiation status and tissue-specific microenvironments.

Biogeographic distribution patterns of algal community in different urban lakes in China: Insights into the dynamics and co-existence.

Urban lake ecosystems are significant for social development, but currently we know little about the geographical distribution of algal community in urban lakes at a large-scale. In this study, we investigated the algal community structure in different areas of urban lakes in China and evaluated the influence of water quality parameters and geographical location on the algal community. The results showed that obvious differences in water quality and algal communities were observed among urban lakes in different geographical areas. Chlorophyta was the dominant phylum, followed by cyanobacteria in all areas. The network analysis indicated that algal community composition in urban lakes of the western and southern area showed more variations than the eastern and northern areas, respectively. Redundancy analysis and structural equation model revealed that nutrients and pH were dominant environmental factors that affected the algal community, and they showed higher influence than that of iron, manganese and COD Mn concentration. Importantly, algal community and density exhibited longitude and latitude relationship. In general, these results provided an ecological insight into large-scale geographical distributions of algal community in urban lakes, thereby having potential applications for management of the lakes.

Comparative analysis of basic helix-loop-helix gene family among Brassica oleracea, Brassica rapa, and Brassica napus.

BACKGROUND: The basic helix-loop-helix (bHLH) is the second largest gene family in the plant, some members play important roles in pistil development and response to drought, waterlogging, cold stress and salt stress. The bHLH gene family has been identified in many species, except for Brassica oleracea and B. napus thus far. This study aims to identify the bHLH family members in B. oleracea, B. rapa and B. napus, and elucidate the expression, duplication, phylogeny and evolution characters of them. RESULT: A total of 268 bHLH genes in B. oleracea, 440 genes in B. napus, and 251 genes in B. rapa, including 21 new bHLH members, have been identified. Subsequently, the analyses of the phylogenetic trees, conserved motifs and gene structures showed that the members in the same subfamily were highly conserved. Most Ka/Ks values of homologous gene were < 1, which indicated that these genes suffered from strong purifying selection for retention. The retention rates of BrabHLH and BolbHLH genes were 51.6 and 55.1%, respectively. The comparative expression patterns between B. rapa and B. napus showed that they had similar expression patterns in the root and contrasting patterns in the stems, leaves, and reproductive tissues. In addition, there were 41 and 30 differential expression bHLH genes under the treatments of ABA and JA, respectively, and the number of down regulation genes was significantly more than up regulation genes. CONCLUSION: In the present study, we identified and performed the comparative genomics analysis of bHLH gene family among B. oleracea, B. rapa and B. napus, and also investigated their diversity. The expression patterns between B. rapa and B. napus shows that they have the similar expression pattern in the root and opposite patterns in the stems, leaves, and reproduction tissues. Further analysis demonstrated that some bHLH gene members may play crucial roles under the abiotic and biotic stress conditions. This is the first to report on the bHLH gene family analysis in B. oleracea and B. napus, which can offer useful information on the functional analysis of the bHLH gene in plants.

SRSF9 selectively represses ADAR2-mediated editing of brain-specific sites in primates.

Adenosine-to-inosine (A-to-I) RNA editing displays diverse spatial patterns across different tissues. However, the human genome encodes only two catalytically active editing enzymes (ADAR1 and ADAR2), suggesting that other regulatory factors help shape the editing landscape. Here, we show that the splicing factor SRSF9 selectively controls the editing of many brain-specific sites in primates. SRSF9 is more lowly expressed in the brain than in non-brain tissues. Gene perturbation experiments and minigene analysis of candidate sites demonstrated that SRSF9 could robustly repress A-to-I editing by ADAR2. We found that SRSF9 biochemically interacted with ADAR2 in the nucleus via its RRM2 domain. This interaction required the presence of the RNA substrate and disrupted the formation of ADAR2 dimers. Transcriptome-wide location analysis and RNA sequencing revealed 1328 editing sites that are controlled directly by SRSF9. This regulon is significantly enriched for brain-specific sites. We further uncovered a novel motif in the ADAR2-dependent SRSF9 binding sites and provided evidence that the splicing factor prevents loss of cell viability by inhibiting ADAR2-mediated editing of genes involved in proteostasis, energy metabolism, the cell cycle and DNA repair. Collectively, our results highlight the importance of SRSF9 as an editing regulator and suggest potential roles for other splicing factors.

Functionality-Independent DNA Encoding of Complex Natural Products.

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late-stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end-products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).

A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing.

CRISPR-Cas9 has emerged as a powerful technology that enables ready modification of the mammalian genome. The ability to modulate Cas9 activity can reduce off-target cleavage and facilitate precise genome engineering. Here we report the development of a Cas9 variant whose activity can be switched on and off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the Cas9 enzyme with the hormone-binding domain of the estrogen receptor (ERT2). The final optimized variant, termed iCas, showed low endonuclease activity without 4-HT but high editing efficiency at multiple loci with the chemical. We also tuned the duration and concentration of 4-HT treatment to reduce off-target genome modification. Additionally, we benchmarked iCas against other chemical-inducible methods and found that it had the fastest on rate and that its activity could be toggled on and off repeatedly. Collectively, these results highlight the utility of iCas for rapid and reversible control of genome-editing function.

SERPINB5 Expression: Association with CCRT Response and Prognostic Value in Rectal Cancer.

Background: Due to the varying characteristics and conflicting outcomes on the overall survival of rectal cancer patients, many studies have been undertaken to determine various prognostic and predictive factors for the mainstay treatment of CCRT followed by surgery. Cancer cell motility contributes to tumor invasion, migration and eventually metastasis. However, the genes associated with cell motility (i.e., GO:0048870) have not been systemically evaluated in rectal cancers. Methods: A comparative analysis of gene expression profiles was applied to the transcriptomic dataset (GSE35452) with a focus on genes associated with cell motility (GO:0048870), where SERPINB5 was recognized as the most significantly up-regulated gene. Tumor samples from 172 primary rectal cancer patients who underwent neoadjuvant CCRT followed by surgical resection were collected. Immunohistochemistry was used to semi-quantitatively assess the expression level of SERPINB5 protein. Statistical analyses of SERPINB5 expression and various clinicopathological features as well as survival were then performed. Results: High immunoreactivity of SERPINB5 was significantly linked to pre- and post-CCRT advanced disease, lymphovascular invasion, and poor response to CCRT (all P ≤ 0.015). SERPINB5 overexpression was not only negatively associated with disease-specific survival (DSS), local recurrence-free survival (LRFS) and metastasis-free survival (MeFS) rates in univariate analyses but also was an independent prognostic factor for DSS and MeFS in rectal cancer patients (all P ≤ 0.043). Conclusion: SERPINB5 may play an important role in rectal cancer progression and response to neoadjuvant CCRT and serve as a novel prognostic factor.

Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction between the GAIN and EGF5 domains accounting for the absence of expression of the GAIN domain itself, although EGF5-GAIN was expressed similarly in the wild-type protein. For HeLa cell adhesion, the GAIN-truncated forms showed dramatically reduced binding affinity. The PNGase F-deglycosylated and GPS mutated forms also exhibited reduced HeLa attachment compared with WT CD97. However, neither N-glycosylation mutagenesis nor auto-proteolysis inhibition caused by N-glycosylation mutagenesis affected CD97-HeLa cell interactions. A comparison of the HeLa binding affinities of PNGase F-digested, GPS-mutated and N-glycosylation-mutated CD97 samples revealed diverse findings, suggesting that the functions of CD97 ECD were complex, and various technologies for function validation should be utilized to avoid single-approach bias when investigating N-glycosylation and auto-proteolysis of CD97. A unique mechanism of concentration-dependent auto-proteolysis of the CD97 EGF1-5 isoform was characterized, suggesting an intermolecular mechanism that is distinct from that of other previously reported CD97 isoforms. The EGF5 and GAIN domains are likely associated with each other as CD97 expression and SAXS data revealed a potential interaction between the two domains. Finally, the GAIN and EGF domains are also important for CD97-HeLa adhesion, whereas N-glycosylation of the CD97 GAIN domain and GPS auto-proteolysis are not required for HeLa cell attachment.